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Effects of cholecystokinin on male copulatory behavior and lordosis behavior in male rats
Physiology & Behavior, Vol. 43, pp. 351-357. Copyright©PergamonPress plc, 1988. Printedin the U.S.A.
0031-9384/88$3.00 + .00
Effects of Cholecystokinin on Male Copulatory Behavior and Lordosis Behavior in Male Rats G E O R G E J. B L O C H , A L E X M. B A B C O C K , R O G E R A. G O R S K I A N D P. E. M I C E V Y C H
D e p a r t m e n t o f A n a t o m y and the Laboratory o f Neuroendocrinology o f the Brain R e s e a r c h Institute U C L A School o f Medicine, Los Angeles, CA 90024
R e c e i v e d 28 D e c e m b e r 1987 BLOCH, G. J., A. M. BABCOCK, R. A. GORSKI AND P. E. MICEVYCH. Effects of cholecystokinin on male copulatory behavior and lordosis behavior in male rats. PHYSIOL BEHAV 43(3) 351-357, 1988.--Because the distribution of cholecystokinin octapeptide (CCK-8) within the hypothalamus and limibc system overlaps with steroid concentrating regions, and because these areas are involved in the regulation of reproductive behaviors, we examined the effects of exogenous CCK-8 on male copulatory behavior and lordosis behavior in the male rat. Peripheral administration of a dose of CCK-8 that altered lordosis behavior in females (3 /xg/kg, intraperitoneal) was ineffective in altering male copulatory behavior in males, either before or after gonadectomy, and was also ineffective in altering lordosis behavior after estrogen priming. In a separate experiment, CCK-8 injected into the lateral ventricle also did not affect male copulatory behavior, but lordosis behavior was increased dramatically after gonadectomy and estrogen priming. Although these results do not answer the question whether CCK-8 is acting to inhibit a neural system that normally suppresses lordosis behavior or is acting to stimulate a facilitatory circuit, these results do indicate the existence of an estrogen sensitive neural substrate in males on which CCK can act to facilitate lordosis behavior. Cholecystokinin
Intraventricular
Gonadectomy
Estrogen
regulation of reproductive behavior. We [5] and others [35] reported that CCK-8 administered intraperitoneally (IP) inhibited lordosis behavior in female rats. In addition, we have reported that, depending on the receptivity of the animal, CCK-8 administered IP can facilitate or inhibit lordosis behavior in the estrogen-primed female and that these effects are not dependent on progesterone [5]. Moreover, the VMH appears to be involved in mediating the inhibitory component of C C K ' s action on lordosis behavior since injection of as little as 4.4 pmoles in this region inhibits estrogen-facilitated lordosis [3]. Because of the sexual dimorphism in the number of CCK immunoreactive cells in the sexually dimorphic circuit [36,39], the apparent change in the number of cells producing CCK in the absence of testosterone [36,51], and the known involvement of these areas in the central nervous system regulation of male reproductive behavior [2, 9, 14, 23, 24, 32, 52], we examined the effects of exogenous CCK-8 on male copulatory behavior and lordosis behavior in the male rat. We report that neither IP nor intraventricular (ICV) administration of CCK-8 significantly alters any component of male copulatory behavior in the intact male. In addition, CCK-8 administered IP does not significantly alter male behavior
CHOLECYSTOKININ octapeptide (CCK-8) is a neuroactive peptide that has a wide distribution in the CNS [55]. Within the hypothalamus and limbic system, this distribution overlaps with steroid concentrating regions that include the medial preoptic area (MPO), bed nucleus of the stria terminalis (BST), medial amygdala (MeA), and ventromedial nucleus of the hypothalamus (VMH) [36, 39, 44, 48, 54]. The volumes of these nuclei are greater in males than in females [22, 26, 33], as is the tissue content of CCK [20, 38, 50] and the number of CCK immunoreactive cells [36,39]. In the VMH, which receives a dense innervation from CCK-8 immunoreactive terminals, the concentration and binding of CCK-8 fibers vary during the estrous cycle [1,36]. These findings imply a role for CCK in neuroendocrine processes, and indeed CCK-8 has been reported to inhibit [56] or to stimulate [29] the release of LH in females, as well as to inhibit the stimulated release of LHRH in vitro from male, ovariectomized, and ovariectomized estrogen-primed rat hypothalami [37]. In addition, gonadectomy of the male reduces the number of CCK immunoreactive cells in the medial preoptic nucleus, BST, and MeA from 2 to 8 times the number in females [36, 39, 51] to female levels [36,51]. Recent evidence has suggested a role of CCK-8 in the
1Supported by NIH grants NS 21220 (P.E.M.) and HD 01182 (R.A.G.). Preliminary reports were given at the Society for Neuroscience and International Congress of the International Society of Psychoneuroendocrinology in 1986 and 1987, respectively.
351
352
BLOCH, BABCOCK, G O R S K I A N D MICEVYCH
following castration. In sharp contrast, gonadectomized estrogen-primed males show a dramatic increase in lordosis behavior following ICV CCK-8. GENERAL METHOD Animals
Animals were 42 male Long-Evans rats obtained from Charles River, Wilmington, MA, at 65 days of age. These rats had Purina lab chow and water available ad lib in a room maintained on a partially reversed light schedule (lights on from 1:30 a.m. to 3:30 p.m.). All rats were given preliminary mating tests in order to select males with proven male copulatory ability. Thirty-six of the 42 males exhibited intromission behavior followed by ejaculation in 2 consecutive tests, and these 36 males were used in the study. Behavioral Testing
All testing sessions began 1.5 hours after lights off and were conducted in Plexiglas mating arenas illuminated with a 25 watt red bulb. Male copulatory behavior was tested by placing the experimental male in the mating arena and then introducing a sexually receptive (stimulus) female ten minutes later. If no intromissions occurred within 10 minutes, a new stimulus female was introduced, and if no intromission occurred during a 20 minute period, the test was terminated. Standard measurements of male behavior were recorded using an Esterline-Angus event recorder [6]: the time from the beginning of the test to the first intromission (intromission latency, IL), the number of mounts or intromissions to ejaculation (mount frequency, MF; intromission frequency, IF), the time from the first intromission to ejaculation (ejaculation latency, EL), the average time between intromissions (intercopulatory interval, I C I = E L / I F ) , and the time from ejaculation to the next intromission (postejaculatory interval, PEI). When ejaculation occurred, the test was terminated after the next intromission. Lordosis behavior was tested as described previously [5]. Briefly, an experienced male was allowed to mount the experimental male 10 times, from which a lordosis quotient (LQ) was calculated (number of lordosis/number of mounts × 100). The male's behavior was recorded as a lordotic response when he displayed an elevation of the perineum and a raising of the head. Peptide Preparation
Sulphated CCK-8 (Bachem, Tustin, CA) was reconstituted with double distilled water, relyophilized in 20 /xg aliquots, and stored desicated at -20°C. Two different vehicles were used: 0.9% saline for IP injections, and an artificial CSF (aCSF) solution for ICV injections (126 mM NaC1, 25 mM Hepes, 6 mM KCI, 1.45 mM CaClz, 1.0 mM NaH2PO4, and 0.88 mM MgSo4; pH, 7,2). The a C S F was passed through a 0.2/zm sterilizing filter (Millipore Corp.) [3]. Fresh CCK-8 solutions were prepared immediately before each testing session. EXPERIMENT 1
The purpose of this experiment was to assess the effects of peripheral administration of CCK-8 on male copulatory behavior and lordosis behavior in males. Method
Fifteen male rats were preselected for male sexual behav-
ior (see General Method section). Since an IP dose of 3 p~g/kg CCK-8 was effective in females [5], this dose was selected for experimental males. Male copulato~ behavior. Ten minutes before each mating test, rats were given an IP injection of 3 p~g/kg sulphated CCK-8 (experimental males) or saline (controls). The first two mating tests were spaced 2 weeks apart and were performed on intact males. These rats were then gonadectomized using ether anesthesia, and a total of 7 postcastrational mating tests were conducted, the first occurring 3 days after castration and the last 50 days after castration. The animals were administered CCK-8 or saline in a crossover design that was used throughout the experiment, and standard measurements of male copulatory behavior were obtained (see General Method section). Data were evaluated for each behavioral measurement by comparing the CCK-8 and saline values over tests 1 and 2 (intact) and 3 through 6 (castrate) using a two-tailed matched-pair t-test. Lordosis behavior. Starting one month after the last test for male behavior, 14 of the gonadectomized males were injected daily at 11:30 a.m. with 5 t~g estradiol benzoate (EB) for 3 days and tested for lordosis behavior on the fourth day (see General Method section.) EB was injected subcutaneously in 0.1 ml sesame oil. Animals received IP injections of 3/zg/kg sulphated CCK-8 (n=7) or saline (n=7) ten minutes before the mating test. Lordosis quotients were calculated and an independent t-test was used to evaluate the data. Results and Discussion
Peripheral administration of CCK-8 did not significantly alter male sexual behavior in the intact males compared with the saline controls (Table 1). Male behavior declined progressively in the 7 mating tests conducted after castration, but there were no significant differences between CCK-8 and vehicle groups in any of the behavioral parameters (Table 1) or in the percentage of animals showing ejaculatory behavior. Ejaculation latencies were reduced in intact males given CCK-8 in the second mating test (441-+32 CCK-8 vs. 575---27 saline, p<0.01, independent t-test). However, no such reduction was observed with CCK-8 in the first test of intact males, nor in the postcastration tests. Moreover, when data were analyzed using matched pairs from the crossover design, ejaculation latencies were not significantly different between CCK-8 and control treatments either before or after castration. Because CCK-8 had no effect following castration, the lack of an effect of CCK-8 on male behavior in the present experiment does not appear to be directly related to the presence of testicular hormones in adulthood, or to the normally high levels of copulatory behavior displayed by intact males which may have masked an effect. Using a higher dose of CCK-8 than we used (5 tzg/rat, IP), Linden et al. [31] also observed no effects on male copulatory behavior. Pfaus et al. [46] reported that males injected peripherally with 8 t~g/kg (subcutaneous) had a reduced ejaculation latency and intromission frequency. Since 8/zg/kg CCK-8 is a considerably higher dose than what is needed to decrease exploratory behaviors [11] and food consumption [12,30] in rats, and since ejaculation latency and intromission frequency can be reduced by "stressful" events such as operative trauma (sham castration) or audiogenic stimulation [15], perhaps the regimen used by Pfaus et al. [46] reduced these behavioral parameters by acting in some stress-related manner. Following estrogen priming, lordosis behavior was also
CCK-8 AND REPRODUCTIVE
BEHAVIORS IN MALES
353
TABLE 1 EFFECTS OF PERIPHERALLY ADMINISTERED CCK-8 (3 g.g/kg) OR SALINE (IP) ON SCORES OF MALE COPULATORY BEHAVIOR* IN INTACT AND GONADECTOMIZED MALE RATS (MEAN - SE) Days Post Castration
Treatment
(N)t
IL 57.9 --- 18 138.6 ± 64
0 (Test 1)
Saline CCK-8
(7) (8)
0 (Test 2)
Saline CCK-8
(7) (7)
3
Saline CCK-8
9
MF
IF
EL
ICI
PEI
8.7 ± 7.3 ±
1 2
15.4 _ 1 11.8 --+ 1
477 678
± 63 ± 143
59.8 --- 12.4 74.0 --+ 15.4
308 ± 52 394 ± 21
22 12
8.7 ± 11.9 ±
1 3
15.4 ± 1 11.6 ± 1.3
575 441
± 27 --- 31
39.3 ± 42.4 ±
358 ± 23 323 ± 20
(7) (7)
53.4 ± 29 148.3 ± 112
8.9 ± 9.1 ±
3.6 2.2
10.9 ± 1 8.9± 1
497 424
± 102 ± 49
52 48
± 11 ± 5
475 ± 476 ±
Saline CCK-8
(4) (6)
209.8 ± 112 255.0 -+ 163
6.3 ± 12.0 ±
3 4
7.0 ± 1 6.3 ± 0.7
293 548
± 73 ± 163
40 94
± 8 ± 35
618 ± 144 547 ± 86
13
Saline CCK-8
(5) (2)
117.8 ± 56 663.0± 605
15.2 - 8 12.5-+ 5
7.0 ± 0.7 9.0 ± 0
493 470
± 207 ± 99
66 47
± 24 --- 6
758 ± 156 578 ± 63
18
Saline CCK-8
(2) (6)
402.0 ± 293 236.0 ± 113
18.0 ± 7 25.0 -+ 9
8.0 ± 1 7.0 ± 1
1194 941
± 116 ± 263
141 205
± 22 ± 79
552 ± 10 464 --- 101
23
Saline CCK-8
(5) (1)
236.0 ± 771.0
22.0 ± 27.0
7.0 --- 1 7.0
624 918
± 206
114 131
± 61
495 ± 100 675
34
Saline CCK-8
(0) (3)
. 291.0 ±
. 9.7 ± 1
. . 884.7 ± 421
82
± 25
499 _
Saline CCK-8
(1) (0)
126
50
63.0 ± 32.6 ±
95
8
. 82
. 24.0 ± 15 20.0
.
.
14.0 .
.
1234 .
88
3.4 3.8
32 40
15
843
.
*IL=intromission latency (sec). M F = m o u n t frequency. IF=intromission frequency. EL =ejaculation latency (sec). ICI=intercopulatory interval (sec). PEI=postejaculatory interval (sec). "~Number of animals displaying ejaculatory pattern. Method n o t significantly d i f f e r e n t b e t w e e n I P C C K - 8 a n d salinei n j e c t e d g r o u p s (saline, LQ=32.3---15; C C K - 8 , L Q = 2 7 . 4 - + 18.2). T h e s e r e s u l t s d e m o n s t r a t e t h a t a n I P d o s e o f C C K - 8 t h a t d r a m a t i c a l l y a l t e r e d l o r d o s i s b e h a v i o r in g o n a d e c t o m i z e d f e m a l e s [5] w a s totally i n e f f e c t i v e in altering lord o s i s in g o n a d e c t o m i z e d m a l e s . EXPERIMENT 2 B e c a u s e t h e n u m b e r o f C C K - 8 i m m u n o r e a c t i v e cells a n d t h e t i s s u e levels o f C C K - 8 in t h e M P O , B N S T , a n d M e A are highly s e x u a l l y d i m o r p h i c w i t h g r e a t e r v a l u e s in m a l e s t h a n in f e m a l e s [20, 35, 37, 38, 49], w e w e r e r e l u c t a n t to c o n c l u d e t h a t this p e p t i d e h a s n o effect in m a l e s . T h e d o s e o f C C K - 8 m i g h t n o t h a v e b e e n sufficient to affect male c o p u l a t o r y beh a v i o r (see [46]) or the peripherally a d m i n i s t e r e d C C K - 8 might n o t h a v e r e a c h e d t h e a p p r o p r i a t e b r a i n a r e a s . S t e r n et al. [53] r e p o r t e d t h a t 3 H - c a e r u l e i n , a d e c a p e p t i d e w i t h struct u r a l a n d p h y s i o l o g i c a l h o m o l o g i e s to C C K - 8 , p r e f e r e n t i a l l y a c c u m u l a t e d in t h e V M H a f t e r p e r i p h e r a l injection. O t h e r s , however, have argued that CCK-8 administered peripherally is a c t i n g v i a p e r i p h e r a l r e c e p t o r s t h a t lie o u t s i d e t h e b l o o d b r a i n b a r r i e r [12]. I n o r d e r to t e s t f u r t h e r t h e h y p o t h e s i s t h a t C C K - 8 is a c t i n g in t h e C N S o f t h e m a l e rat to m o d u l a t e r e p r o d u c t i v e b e h a v i o r , we a d m i n i s t e r e d C C K - 8 i n t r a v e n tricularly ( I C V ) a n d studied its effects o n male c o p u l a t o r y and lordosis behaviors.
T w e n t y - o n e adult male rats w e r e p r e s e l e c t e d for male s e x u a l b e h a v i o r (see G e n e r a l M e t h o d section). O n e w e e k following p r e s e l e c t i o n tests, rats w e r e a n e s t h e t i z e d w i t h 3 ml/kg E q u i t h e s i n (0.85 g c h l o r a l h y d r a t e , 0.21 g s o d i u m p e n t o b a r b i t a l / 2 0 ml) a n d u n i l a t e r a l guide c a n n u l a e (22 ga) w e r e surgically i m p l a n t e d ; t h e tips w e r e l o c a t e d 1.0 m m a b o v e t h e lateral v e n t r i c l e , a n d c a n n u l a e w e r e a n c h o r e d w i t h skull s c r e w s . R a t s w e r e a l l o w e d o n e w e e k to r e c o v e r b e f o r e b e h a v i o r a l testing. D u r i n g i n j e c t i o n s , r a t s w e r e r e m o v e d f r o m t h e i r c a g e s a n d lightly r e s t r a i n e d . A 30 gauge wire s t y l e t t h a t w a s p r e s e n t in t h e guide c a n n u l a w a s r e m o v e d a n d rep l a c e d w i t h a n i n j e c t i o n c a n n u l a t h a t e x t e n d e d 1.0 m m bey o n d t h e guide c a n n u l a . I n j e c t i o n c a n n u l a e w e r e c o n n e c t e d with p o l y e t h y l e n e tubing to a h a n d d r i v e n microsyringe. Intrav e n t r i c u l a r i n j e c t i o n s o f 5.0/xl w e r e m a d e o v e r a o n e m i n u t e p e r i o d a n d t h e i n j e c t o r r e m a i n e d in place for a n a d d i t i o n a l 30 s e c o n d s to allow for diffusion o f solution f r o m t h e tip. I n o r d e r to verify c o r r e c t p l a c e m e n t o f c a n n u l a e , rats w e r e g i v e n a n o v e r d o s e o f a n e s t h e t i c , g i v e n a 3 . 0 / x l injection o f india ink b y i n s e r t i n g t h e i n j e c t i o n c a n n u l a into the guide c a n n u l a , t h e n p e r f u s e d i n t r a c a r d i a l l y w i t h 0.9% saline foll o w e d b y 10% n o r m a l formalin. B r a i n s w e r e c u t w i t h a r a z o r b l a d e in the c o r o n a l p l a n e , a n d p l a c e m e n t w a s verified b y observing the stained ventricular system. One rat had an i n c o r r e c t c a n n u l a p l a c e m e n t ; the d a t a f r o m this a n i m a l w e r e n o t u s e d in t h e study.
354 M a l e c o p u l a t o r y b e h a v i o r . CCK-8 was administered 10 minutes before behavioral testing. Six males received 50 ng CCK-8, eight received 500 ng CCK-8, and six were injected with the aCSF vehicle (see General Method section). Standard measures of male sexual behavior were obtained as described in the General Method section, and data were evaluated using analysis of variance and Duncan's Multiple Range Test. L o r d o s i s b e h a v i o r . Five days after the male copulatory test, the males were gonadectomized using ether anesthesia. Starting two weeks after gonadectomy, the rats were given four mating tests, each separated by 10 days. In order to safeguard against any bias in treatment, CCK-8 or a C S F treatments were assigned randomly among the animals for each test. Animals were primed with 5 tzg EB/day x 3 days for the first 3 mating tests, and the dose was increased to 30 /zg EB/day x 3 days for the fourth test in order to study the effects of CCK-8 in highly receptive males. Intraventricular injections were performed in the same manner as described for the male copulatory tests, but because of the long period of implantation, we anticipated that some cannulae assembly would eventually become loosely attached to the skull and therefore studied only 2 groups: vehicle (aCSF) and 500 ng CCK-8. Lordosis quotients were obtained as described in the General Method section, and these were evaluated by 2-way analysis of variance (treatment by testing session). Because there was a significant effect of treatment, mating tests were evaluated separately using Bonferroni's method of simultaneous inference [40]. Results and Discussion
Twenty of the 21 rats had histologically confirmed injection sites within the lateral ventricle. During the course of the experiment, the cannulae assembly in 7 of the 20 males became loosely attached to the skull. When this occurred, the animal was removed from further study. All 20 males were studied in the male copulatory test, 18 were studied in the first lordosis test, and this number was further reduced to 13 by the final lordosis test. As in Experiment 1, CCK-8 did not significantly alter any component of male sexual behavior when compared to vehicle controls. Measurements of IL, MF, ICI, and PEI were quite similar among the 0 ng, 50 ng, and 500 ng CCK-8 groups (n =6, 6, and 8, respectively). I F and E L were reduced in the 50 ng and 500 ng CCK-8 males, but individual values were quite variable and these effects were not statistically significant. (IF: 11.2-+1.5 in the 0 ng CCK-8 group, and 6.8-+1.2 and 8.3+1.6 in the 50 ng and 500 ng groups, respectively. EL: 825-+226 seconds in the 0 ng CCK-8 group, and 527-+189 and 474+14 in the 50 ng and 500 ng groups). All animals showed ejaculatory behavior during the behavioral test. Following gonadectomy and estrogen priming, animals receiving 500 ng CCK-8 ICV showed dramatically higher L Q ' s than aCSF controls. There was a significant treatment effect, F=16.94, p<0.001, but there was no significant effect of testing session and no significant treatment x testing session interaction. Comparison between CCK-8 and aCSF groups revealed that CCK-8 ICV increased L Q ' s significantly in tests 2 and 3 (Fig. 1). The results of this experiment demonstrate that injection of 50 ng or 500 ng CCK-8 ICV had no significant effects on male copulatory behavior in intact males. These results may imply that male rats are refractive to the effects of CCK-8.
BLOCH, BABCOCK, GORSKI A N D MICEVYCH
100 -
i
9080~- 7 0 z w
60-
O
D 50
o
--- 4 0
o~ 3o n~ o ~ 20
lO 0 DALLYEB DOSE:
//// ////
U// I///
8 TEST I 5#g
9 TEST 2 5~Q
. . . .
! ......
TEST 3 5~g
??5
6
TEST 4 30/Jg
FIG. 1. Effects of intracerebroventricular administration of 500 ng CCK-8 (clear bars) or artificial CSF (vehicle) on lordosis behavior in estrogen-primed males gonadectomized in adulthood. Animals were given 5 tzg EB/day x 3 days before Tests 1-3 and 30 p.g EB/day x3 days before Test 4. Each test was separated by 10 days. *p<0.02 vs. CSF treated males.
However, the dramatic increase in the LQ of gonadectomized estrogen-primed male rats (see Fig. 1) indicates that 500 ng CCK-8 is capable of affecting reproductive behavior in males. As expected, the LQ of the a C S F controls was quite high in the fourth lordosis test, which was conducted after estrogen priming with 30/zg EB. This is in agreement with previous reports showing that the LQ of gonadectomized, EB-primed males increases with repeated tests at a given dose of estrogen priming, or with higher doses of EB [15,17]. In sharp contrast with our findings in females given IP CCK-8 [5] or CCK-8 within the VMH [3], there was no indication in the present study of a reduced LQ in males after ICV injection. Indeed, under conditions of increasing behavioral receptivity the LQ was still higher in the males treated with ICV CCK-8 than in the C S F controls (Fig. 1, test 4). GENERAL DISCUSSION CCK-8 failed to alter any parameter of male copulatory behavior after peripheral (IP) injections in intact or gonadectomized male rats, or after central (ICV) injections. In sharp contrast, ICV CCK-8 significantly and dramatically increased the frequency of lordotic responses in gonadectomized, estrogen-primed males. Several lines of evidence indicate that the lack of a CCK-8 effect on male behavior is not due to an inability of ICV CCK-8 to influence the CNS. Firstly, a dose of CCK-8 that did not alter male copulatory behavior did facilitate lordosis behavior dramatically in the same males. Secondly, the 500 ng dose that we used is approximately 100 fold higher than the effective dose to inhibit L H secretion when injected within the third ventricle of the female rat [56] and between 10 and 100 times higher than the dose required to reduce LQ scores when administered within the VMH of female rats [3]. When administered ICV, the doses of CCK-8 required to suppress feeding and exploratory behaviors are higher than we used [11,49], so it is possible that the dose we used was insufficient to influence male behavior. Dornan and Malsbury [19], however, have reported that 200 ng of CCK-8 injected into the preoptic-anterior hypothalamic area of males failed to affect male copulatory behavior, indicating that injection of a relatively high dose of CCK-8 into a region
CCK-8 A N D R E P R O D U C T I V E B E H A V I O R S IN M A L E S
355
that is important for the expression of male copulatory behavior [2, 14, 24, 32, 52] was also ineffective. The dramatic facilitatory effect of ICV CCK-8 on lordosis behavior in males is especially interesting. In contrast to females, males gonadectomized in adulthood show relatively low levels of lordosis behavior following estrogen or combined estrogen/progesterone treatment [10,16]. The relatively low level of lordosis behavior in males is due to the influence of testicular hormones exerted during the neonatal period; gonadectomy during the neonatal period but not at a later time results in a male that shows a level of receptivity in adulthood equivalent to that displayed by normal females (for review, see [21]). Lordosis behavior can also be increased by experimental manipulation of males in adulthood. When estrogen is administered in high daily doses to male gonadectomized as adults, the frequency of lordotic responses approaches that of females, although much higher doses o f estrogen are needed for similar effects [16,17]. It is therefore possible that substrates exist within the male brain that influence lordosis behavior in a manner similar to that proposed for the female. Indeed, although the dose is much higher than required in a female, estrogen implants within the VMH also increase lordosis behavior in male rats [18]. As in the female, lesions of the VMH reduce lordosis behavior of the male [8] while lesions of the POA increase lordosis behavior [25]. Because lesions of the lateral septum [41,42] or anterior roof deafferentation, which cuts the connections between the septum and the preoptic area [59], also increase lordosis behavior in males, it appears that the behaviorally differentiating action of testicular hormones in the neonatal period may be due, in part, to the development of a neural system that suppresses lordosis in the normal male [25]. We have shown that CCK-8 itself will not induce lordosis behavior in females [5] and males (unpublished observation) but modifies only an estrogen-primed system. Thus, the relatively rapid effects of CCK-8, occurring within 10 minutes following its administration ICV, could be exerted through an interaction with cells in one or more areas of the brain, but only after estrogen has been allowed to initiate de novo synthesis of proteins or other substrates essential for lordosis behavior [28, 34, 58]. Because increased sexual receptivity in estrogen-primed female rats can be accomplished by giving progesterone or " p r o g e s t e r o n e substitutes" that include cholinergic agonists, serotonergic agonists and antagonists, dopaminergic agonists and antagonists, ovarian and adrenal hormones, and a variety of peptides (see [13, 27, 57]), this has led to speculation that a "nonspecific" mechanism may be involved [57]. However, the male rat differs from the female in that lordosis behavior in the estrogen-primed normal [10,17] or septal-lesioned [41] male is not further increased by progesterone. Moreover, the facilitation of lordosis behavior following peripheral administration of CCK-8 in lowreceptive, ovariectomized, estrogen-primed female rats is also observed after adrenalectomy, indicating that progesterone is not required for this effect in females [5]. Thus, it appears unlikely that CCK-8 would be acting on this system in the male. We have proposed that CCK-8 is acting within the hypothalamus of the female to inhibit the effects on lordosis behavior that are attributed to that site [3]. The regions of the
hypothalamus that appear to be most important for the regulation of the lordosis behavior in estrogen-primed female and male rats are the VMH and MPO [7, 8, 25, 43, 45, 47]. Lordosis behavior of the female rat appears to be under the dual control of these regions. According to this concept, estrogen stimulates cells of the V M H to increase lordosis behavior of the female and simultaneously estrogen acts in the POA to remove preoptic inhibition of this behavior [4,7]. Thus, the V M H is a facilitatory site for lordosis behavior in the estrogen-primed female (see [43] for review), and CCK-8 could be acting to inhibit this action and thereby attenuate this behavior. Similarly, CCK-8 could be acting to inhibit the inhibitory action of the MPO on lordosis behavior in the female [7, 45, 47] and thereby increase this behavior. As discussed previously [3], this model can explain the types of complex results in the female that we have reported after peripheral administration of CCK-8 [5]. In the estrogenprimed female rat, CCK-8 administered into the V M H decreases lordosis behavior [3], while preliminary results in our laboratory indicate that CCK-8 administered into the MPO facilitates this behavior (Dornan, Bloch and Micevych, unpublished). In the present experiment, lordosis behavior was not reduced in males administered CCK-8 ICV under conditions of high receptivity, a result that we have observed after peripheral [5] or VMH [3] administration of this peptide in females. One explanation for this difference in response to CCK-8 between males and females is that the ICV administered CCK-8 in the present study did not perfuse the VMH whereas the peripheral (and VMH) application in females did reach this brain area. Indeed, peripherally administered CCK-8 and injections of CCK-8 into the VMH may be acting on the same site [53]. A more likely explanation is that the CCK-sensitive neural circuits which regulate lordosis behavior are sexually dimorphic. There are several sex differences within the regions of the hypothalamus and limbic system that contain CCK-8 (see Introduction). We are therefore testing the possibility that the neural substrate responsible for the initiation of lordosis behavior is different in males and females. In the estrogen-primed male, preliminary results indicate that, as in the female, CCK-8 administered into the MPO has a facilitatory effect on lordosis behavior (Bloch et al., unpublished), and we are also studying males that will receive CCK-8 in the VMH. By comparing the doses of CCK-8 that are needed to produce alterations of lordosis behavior in the male and the female, we will test the hypothesis that there are site-specific sex differences in the lordosis response to CCK-8. In summary, we have demonstrated that peripheral administration of a dose of CCK-8 that is effective in altering lordosis behavior in females does not alter either male or female reproductive behavior in males. Although central administration of CCK-8 also did not affect male copulatory behavior, lordosis behavior was dramatically increased in these males after gonadectomy and estrogen priming. These results indicate that exogenous CCK-8 is acting in the male either to inhibit a neural system that normally suppresses lordosis behavior, or is acting directly on neural substrates that facilitate lordosis behavior. In addition, these results imply that there may be a significant sex difference in how CCK-responsive circuits are organized within the brain to regulate lordosis behavior.
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REFERENCES 1. Akesson, T. R.; Mantyh, P. W.; Mantyh, C. R.; Matt, D. W.; Micevych, P. E. Estrous cyclicity of 125-1-CCK octapeptide binding in the ventromedial nucleus: evidence for down modulation by estrogen. Neuroendocrinology 45:257-262; 1986. 2. Arendash, G. W.; Gorski, R. A. Effects of discrete lesions of the sexually dimorphic nucleus of the preoptic area or other medial preoptic regions on the sexual behavior of male rats. Brain Res. Bull. 10:147-154; 1983. 3. Babcock, A. M.; Bloch, G. J.; Micevych, P. E. Injections of cholecystokinin into the ventromedial hypothalamic nucleus inhibit lordosis behavior in the rat. Physiol. Behav., in press; 1988. 4. Bartield, R. J.; Chen, J. J. Activation of estrous behavior in ovariectomized rats by intracerebral implants of estradiol benzoate. Endocrinology 101:1716-1725; 1977. 5. Bloch, G. J.; Babcock, A. M.; Gorski, R. A.; Micevych, P. E. Cholecystokinin stimulates and inhibits lordosis behavior in female rats. Physiol. Behav. 39:217-224; 1987. 6. Bloch, G. J.; Davidson, J. M. Effects of adrenalectomy and experience on postcastration sex behavior in the male rat. Physiol. Behav. 3:461-465; 1968. 7. Bueno, J.; Pfaff, D. W. Single unit recording in hypothalamus and preoptic area of estrogen-treated and untreated ovariectomized female rats. Brain Res. 101:67-78; 1976. 8. Chateau, D.; Chabli, A.; Aron, C. Effects of ventromedial nucleus lesions on the display of lordosis behavior in the male rat. Interactions with facilitory effects of male urine. Physiol. Behav. 39:341-345; 1987. 9. Christenson, L. W.; Nance, D. M.; Gorski, R. A. Effects of hypothalamic and preoptic lesions on reproductive behavior in male rats. Brain Res. Bull. 2:137-141; 1977. 10. Clemens, L. G.; Shryne, J.; Gorski, R. A. Androgen and development of progesterone responsiveness in male and female rats. Physiol. Behav. 5:673-678; 1970. 11. Crawley, J. N.; Hays, S. E.; O'Donahue, T. L.; Paul, S. M.; Goodwin, F. K. Neuropeptide modulation of social and exploratory behaviors in laboratory rodents. Peptides 2:Suppl 1, 123129; 1981. 12. Crawley, J. N.; Rojas-Ramirez, J. A.; Mendelson, W. B. The role of central and peripheral cholecystokinin in mediating appetitive behaviors. Peptides 3:535-538; 1982. 13. Crowley, W. R.; Zemlan, F. P. The neurochemical control of mating behavior. In: Adler, N. T., ed. Neuroendocrinology of reproduction. New York: Plenum Press, 1981:451-484. 14. Davidson, J. M. Activation of the male rat's sexual behavior by intracerebral implantation of adrogen. Endocrinology 79:783794; 1966. 15. Davidson, J. M. Hormonal control of sexual behavior in adult rats. In: Raspe, G., ed. Advances in the bioscieneces. (Schering symposium on endocrinology). New York: Pergamon Press, 1967:11%141. 16. Davidson, J. M. Effects of estrogen on the sexual behavior of male rats. Endocrinology 84:1365--1372; 1969. 17. Davidson, J. M.; Bloch, G. J. Neuroendocrine aspects of male reproduction. Biol. Reprod. 1:67-92; 1969 18. Davis, P. G.; Barfield, R. J. Activation of feminine sexual behavior in castrated male rats by intrahypothalamic implants of estradiol benzoate. Neuroendocrinology 28:228-233; 1979. 19. Dornan, W. A.; Malsbury, C. W. The effects of medial preoptic area injections of substance P and cholecystokinin on male rat sexual behavior. Ann. Conf. Sex. Behav. 19:43; 1987. 20. Frankfurt, M.; Siegel, R. A.; Sim, I.; Wuttke, W. Cholecystokinin and Substance P concentrations in discrete areas of the rat brain: sex differences. Brain Res, 358:53-58; 1985. 21. Gorski, R. A. The neuroendocrine regulation of sexual behavior. In: Newton, G.; Riesen A. H., eds. Advances in psychobiology. New York: John Wiley & Sons, 1974:1-58.
22. Gorski, R. A.; Harlan, R. E.; Jacobson, C. D.; Shryne, J. E.; Southam, A. M. Evidence for the existence of a sexually dimorphic nucleus in the preoptic area of the rat. J. Comp. Neurol. 193:52%539; 1980. 23. Harris, V. S.; Sachs, B. D. Copulatory behavior in male rats following amygdaloid lesions. Brain Res. 86:514-518; 1975. 24. Heimer, L. ; Larsson, K. Impairment of mating behavior in male rats following lesions of the preoptic-anterior hypothalamic continuum. Brain Res. 3:248-263; 1966/1967. 25. Hennessey, A. C.; Wallen, K. ; Edwards, D. A. Preoptic lesions increase the display of lordosis by male rats. Brain Res. 370: 21-28; 1986. 26. Hines, M.; Davis, F. C.; Coquelin, A.; Goy, R. W.; Gorski, R. A. Sexually dimorphic regions in the medial preoptic area and the bed nucleus of the stria terminalis of the guinea pig brain: a description and an investigation of their relationship to gonadal steroids in adulthood. J. Neurosci. 5:40-47; 1985. 27. Hunter, A. J.; Hole, D. R.; Wilson, C. A. Studies into the dual effects of serotonergic pharmacological agents on female sexual behavior in the rat: Preliminary evidence that endogenous 5HT is stimulatory. Pharmacol. Biochem. Behav. 22:5-13, 1985. 28. Jones, K. J.; Chikaraishi, D. M.; Harrington, C. A.; McEwen, B. S.; Pfaff, D. W. In situ hybridization detection of estradiolinduced changes in ribosomal RNA levels in rat brain. Mol. Brain Res. 1:145-152; 1986. 29. Kimura, F.; Hashimoto, R.; Kawakami, M. The stimulatory effect of cholecystokinin implanted in the medial preoptic area on luteinizing hormone secretion in the ovariectomized estrogen-primed rat. Endocrinol. Jpn. 30:305-309; 1983. 30. Kraly, F. S. A diurnal variation in the satiating potency of cholecystokinin in the rat. Appetite: J. Intake Res. 2: 177-191; 1981. 31. Linden, A.; Hansen, S.; Bednar, I.; Forsberg, G.; Srdersten, P. ; Uvnas-Moberg, K. Sexual activity increases plasma concentrations of cholecystokinin octapeptide and offsets hunger in male rats. J. Endocrinol. 115:91-95; 1987. 32. Lisk, R. D. Copulatory ability of the male rat following placement of medial preoptic-anterior hypothalamic lesions. Exp. Brain Res. 5:306-313; 1968. 33. Matsumoto, A.; Arai, Y. Sex difference in volume of the ventromedial nucleus of the hypothalamus in the rat. Endocrinol. Jpn. 30:277-280; 1983. 34. Meisel, R. L.; Pfaff, D. W. RNA and protein synthesis inhibitors: Effects on sexual behavior in female rats. Brain Res. Bull. 12:187-193; 1984. 35. Mendelson, S. D.; Gorzalka, B. B. Cholecystokinin-octapeptide produces inhibition of lordosis in the female rat. Pharmacol. Biochem. Behav. 21:755-759; 1984. 36. Micevych, P. E.; Akesson, T. R.; Elde, R. P. The distribution of cholecystokinin immunoreactive cell bodies in the male and female rat. II. Bed nucleus and amygdaloid complex. J. Comp. Neurol., in press; 1988. 37. Micevych, P. E.; Babcock, A. M.; Matt, D. W.; Lu, J. K. H. Cholecystokinin inhibits k+ stimulated LHRH release from male and female rat hypothalami in vitro. Soc. Neurosci. Abstr. 12:1415; 1986. 38. Micevych, P. E.; Matt, D. W.; Go, V. W. Brain distribution of CCK, sP and bombesin in male and female rats during the estrous cycle. Exp. Neurol., in press; 1988. 39. Micevych, P. E.; Park, S. S.; Akesson, T. R.; Elde, R. Distribution of cholecystokinin-immunoreactive cell bodies in the male and female rat: I. Hypothalamus. J. Comp. Neurol. 255:124-136; 1987. 40. Moses, L. E. Think and explain with statistics. Reading, MA: Addison-Wesley; 1986. 41. Nance, D. M.; Shryne, J.; Gorski, R. A. Septal lesions: effects on lordosis behavior and pattern of gonadotropin release. Horm. Behav. 5:73-81; 1974.
CCK-8 AND REPRODUCTIVE
BEHAVIORS IN MALES
42. Nance, D. M.; Shryne, J. ; Gorski, R. A. Facilitation of female sexual behavior in male rats by septal lesions: an interaction with estrogen. Horm. Behav. 6:289-299; 1975. 43. Pfaff, D. W. Impact of estrogens on hypothalamic nerve cells: ultrastructural, chemical, and electrical effects. Recent Prog. Horm. Res. 39:127-179; 1981. 44. Pfaff, D. W.; Keiner, M. Atlas of estradiol-concentrating cells in the central nervous system of the female rat. J. Comp. Neurol. 151:121-158; 1974. 45. Pfaff, D. W.; Sakuma, Y. Facilitation of the lordosis reflex of female rats from the ventromediai nucleus of the hypothalamus. J. Physiol. 288:189-202; 1979. 46. Pfaus, J. G.; Phillips, A. G. Cholecystokinin facilitates ejaculation in male rats: blockade with progiumide and apomorphine. Eur. J. Pharmacol. 141:331-338; 1987. 47. Powers, B.; Valenstein, E. S. Sexual receptivity: facilitation by medial preoptic lesions in female rats. Science 175:1003-1005; 1972. 48. Sar, M. ; Stumpf, W. E. Distribution of androgen-concentrating neurons in rat brain. In: Stumpf, W. E.; Grant, L. D., eds. Anatomical neuroendocrinology. Basel: Karger, 1975:120-133. 49. Schick, R. R.; Yaksh, T. L.; Go, V. L. W. Intracerebroventricular injections of cholecystokinin octapeptide suppress feeding in rats--pharmacological characterization of this action. Regul. Pept. 14:277-291; 1986. 50. Siegel, R. A.; Frankfurt, M.; Pahnke, U. ; Wuttke, W. Cholecystokinin in discrete rat brain regions as a function of sex and estrous cycle. Ann. NY Acad. Sci 448:651-653; 1985. 51. Simerly, R. B.; Swanson, L. W. Castration reversibly alters levels of cholecystokinin immunoreactivity within cells of three interconnected sexually dimorphic forebrain nuclei in the rat. Proc. Natl. Acad. Sci. USA 84:2087-2091; 1987.
357 52. Soulairac, A.; Soulairac, M.-L. Modifications due comportement exuel et du tractus genital du rat male apres lesions hypothalamiques. Comp. R. Soo. Biol. 150:1097-1100; 1956. 53. Stern, J. J.; Cudillo, C. A. ; Kruper, J. Ventromedial hypothalamus and short-term feeding suppression by caernlein in male rats. J. Comp. Physiol. Psychol. 90:484-490; 1976. 54. Stumpf, W. E.; Sar, M.; Keefer, D. A. Atlas of estrogen target cells in rat brain. In: Stumpf, W. E.; Grant, L. D., eds. Anatomical neuroendocrinology. Basel: Karger, 1975:104--119. 55. Vanderhaeghen, J. J. Neuronal cholecystokinin, in G A B A and neuropeptides in the CNS, Part I. In: Bjorklund, A.; Hokfelt, T., eds. Handbook of chemical neuroanatomy. New York: Elsevier, 1985:406--432. 56. Vijayan, E.; Samson, W. K.; McCann, S. M. In vivo and in vitro effects of cholecystokinin on gonadotropin, prolactin, growth hormone and thyrotropin release in the rat. Brain Res. 172:295-302; 1979. 57. Whalen, R. E.; Lauber, A. H. Progesterone substitutes: cGMP mediation. Neurosci. Biobehav. Rev. 10:47-53; 1986. 58. Yahr, P.; Ulibarri, C. Estrogen induction of sexual behavior in female rats and synthesis of polyadenylated messenger RNA in the ventromedial nucleus of the hypothalamus. Mol. Brain Res. 1:153-165; 1986. 59. Yamanouchi, K.; Arai, Y. Lordosis behavior in male rats: effect of deafferentation in the preoptic area and hypothalamus. J. Endocrinol. 76:381-382; 1978.
0031-9384/88$3.00 + .00
Effects of Cholecystokinin on Male Copulatory Behavior and Lordosis Behavior in Male Rats G E O R G E J. B L O C H , A L E X M. B A B C O C K , R O G E R A. G O R S K I A N D P. E. M I C E V Y C H
D e p a r t m e n t o f A n a t o m y and the Laboratory o f Neuroendocrinology o f the Brain R e s e a r c h Institute U C L A School o f Medicine, Los Angeles, CA 90024
R e c e i v e d 28 D e c e m b e r 1987 BLOCH, G. J., A. M. BABCOCK, R. A. GORSKI AND P. E. MICEVYCH. Effects of cholecystokinin on male copulatory behavior and lordosis behavior in male rats. PHYSIOL BEHAV 43(3) 351-357, 1988.--Because the distribution of cholecystokinin octapeptide (CCK-8) within the hypothalamus and limibc system overlaps with steroid concentrating regions, and because these areas are involved in the regulation of reproductive behaviors, we examined the effects of exogenous CCK-8 on male copulatory behavior and lordosis behavior in the male rat. Peripheral administration of a dose of CCK-8 that altered lordosis behavior in females (3 /xg/kg, intraperitoneal) was ineffective in altering male copulatory behavior in males, either before or after gonadectomy, and was also ineffective in altering lordosis behavior after estrogen priming. In a separate experiment, CCK-8 injected into the lateral ventricle also did not affect male copulatory behavior, but lordosis behavior was increased dramatically after gonadectomy and estrogen priming. Although these results do not answer the question whether CCK-8 is acting to inhibit a neural system that normally suppresses lordosis behavior or is acting to stimulate a facilitatory circuit, these results do indicate the existence of an estrogen sensitive neural substrate in males on which CCK can act to facilitate lordosis behavior. Cholecystokinin
Intraventricular
Gonadectomy
Estrogen
regulation of reproductive behavior. We [5] and others [35] reported that CCK-8 administered intraperitoneally (IP) inhibited lordosis behavior in female rats. In addition, we have reported that, depending on the receptivity of the animal, CCK-8 administered IP can facilitate or inhibit lordosis behavior in the estrogen-primed female and that these effects are not dependent on progesterone [5]. Moreover, the VMH appears to be involved in mediating the inhibitory component of C C K ' s action on lordosis behavior since injection of as little as 4.4 pmoles in this region inhibits estrogen-facilitated lordosis [3]. Because of the sexual dimorphism in the number of CCK immunoreactive cells in the sexually dimorphic circuit [36,39], the apparent change in the number of cells producing CCK in the absence of testosterone [36,51], and the known involvement of these areas in the central nervous system regulation of male reproductive behavior [2, 9, 14, 23, 24, 32, 52], we examined the effects of exogenous CCK-8 on male copulatory behavior and lordosis behavior in the male rat. We report that neither IP nor intraventricular (ICV) administration of CCK-8 significantly alters any component of male copulatory behavior in the intact male. In addition, CCK-8 administered IP does not significantly alter male behavior
CHOLECYSTOKININ octapeptide (CCK-8) is a neuroactive peptide that has a wide distribution in the CNS [55]. Within the hypothalamus and limbic system, this distribution overlaps with steroid concentrating regions that include the medial preoptic area (MPO), bed nucleus of the stria terminalis (BST), medial amygdala (MeA), and ventromedial nucleus of the hypothalamus (VMH) [36, 39, 44, 48, 54]. The volumes of these nuclei are greater in males than in females [22, 26, 33], as is the tissue content of CCK [20, 38, 50] and the number of CCK immunoreactive cells [36,39]. In the VMH, which receives a dense innervation from CCK-8 immunoreactive terminals, the concentration and binding of CCK-8 fibers vary during the estrous cycle [1,36]. These findings imply a role for CCK in neuroendocrine processes, and indeed CCK-8 has been reported to inhibit [56] or to stimulate [29] the release of LH in females, as well as to inhibit the stimulated release of LHRH in vitro from male, ovariectomized, and ovariectomized estrogen-primed rat hypothalami [37]. In addition, gonadectomy of the male reduces the number of CCK immunoreactive cells in the medial preoptic nucleus, BST, and MeA from 2 to 8 times the number in females [36, 39, 51] to female levels [36,51]. Recent evidence has suggested a role of CCK-8 in the
1Supported by NIH grants NS 21220 (P.E.M.) and HD 01182 (R.A.G.). Preliminary reports were given at the Society for Neuroscience and International Congress of the International Society of Psychoneuroendocrinology in 1986 and 1987, respectively.
351
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BLOCH, BABCOCK, G O R S K I A N D MICEVYCH
following castration. In sharp contrast, gonadectomized estrogen-primed males show a dramatic increase in lordosis behavior following ICV CCK-8. GENERAL METHOD Animals
Animals were 42 male Long-Evans rats obtained from Charles River, Wilmington, MA, at 65 days of age. These rats had Purina lab chow and water available ad lib in a room maintained on a partially reversed light schedule (lights on from 1:30 a.m. to 3:30 p.m.). All rats were given preliminary mating tests in order to select males with proven male copulatory ability. Thirty-six of the 42 males exhibited intromission behavior followed by ejaculation in 2 consecutive tests, and these 36 males were used in the study. Behavioral Testing
All testing sessions began 1.5 hours after lights off and were conducted in Plexiglas mating arenas illuminated with a 25 watt red bulb. Male copulatory behavior was tested by placing the experimental male in the mating arena and then introducing a sexually receptive (stimulus) female ten minutes later. If no intromissions occurred within 10 minutes, a new stimulus female was introduced, and if no intromission occurred during a 20 minute period, the test was terminated. Standard measurements of male behavior were recorded using an Esterline-Angus event recorder [6]: the time from the beginning of the test to the first intromission (intromission latency, IL), the number of mounts or intromissions to ejaculation (mount frequency, MF; intromission frequency, IF), the time from the first intromission to ejaculation (ejaculation latency, EL), the average time between intromissions (intercopulatory interval, I C I = E L / I F ) , and the time from ejaculation to the next intromission (postejaculatory interval, PEI). When ejaculation occurred, the test was terminated after the next intromission. Lordosis behavior was tested as described previously [5]. Briefly, an experienced male was allowed to mount the experimental male 10 times, from which a lordosis quotient (LQ) was calculated (number of lordosis/number of mounts × 100). The male's behavior was recorded as a lordotic response when he displayed an elevation of the perineum and a raising of the head. Peptide Preparation
Sulphated CCK-8 (Bachem, Tustin, CA) was reconstituted with double distilled water, relyophilized in 20 /xg aliquots, and stored desicated at -20°C. Two different vehicles were used: 0.9% saline for IP injections, and an artificial CSF (aCSF) solution for ICV injections (126 mM NaC1, 25 mM Hepes, 6 mM KCI, 1.45 mM CaClz, 1.0 mM NaH2PO4, and 0.88 mM MgSo4; pH, 7,2). The a C S F was passed through a 0.2/zm sterilizing filter (Millipore Corp.) [3]. Fresh CCK-8 solutions were prepared immediately before each testing session. EXPERIMENT 1
The purpose of this experiment was to assess the effects of peripheral administration of CCK-8 on male copulatory behavior and lordosis behavior in males. Method
Fifteen male rats were preselected for male sexual behav-
ior (see General Method section). Since an IP dose of 3 p~g/kg CCK-8 was effective in females [5], this dose was selected for experimental males. Male copulato~ behavior. Ten minutes before each mating test, rats were given an IP injection of 3 p~g/kg sulphated CCK-8 (experimental males) or saline (controls). The first two mating tests were spaced 2 weeks apart and were performed on intact males. These rats were then gonadectomized using ether anesthesia, and a total of 7 postcastrational mating tests were conducted, the first occurring 3 days after castration and the last 50 days after castration. The animals were administered CCK-8 or saline in a crossover design that was used throughout the experiment, and standard measurements of male copulatory behavior were obtained (see General Method section). Data were evaluated for each behavioral measurement by comparing the CCK-8 and saline values over tests 1 and 2 (intact) and 3 through 6 (castrate) using a two-tailed matched-pair t-test. Lordosis behavior. Starting one month after the last test for male behavior, 14 of the gonadectomized males were injected daily at 11:30 a.m. with 5 t~g estradiol benzoate (EB) for 3 days and tested for lordosis behavior on the fourth day (see General Method section.) EB was injected subcutaneously in 0.1 ml sesame oil. Animals received IP injections of 3/zg/kg sulphated CCK-8 (n=7) or saline (n=7) ten minutes before the mating test. Lordosis quotients were calculated and an independent t-test was used to evaluate the data. Results and Discussion
Peripheral administration of CCK-8 did not significantly alter male sexual behavior in the intact males compared with the saline controls (Table 1). Male behavior declined progressively in the 7 mating tests conducted after castration, but there were no significant differences between CCK-8 and vehicle groups in any of the behavioral parameters (Table 1) or in the percentage of animals showing ejaculatory behavior. Ejaculation latencies were reduced in intact males given CCK-8 in the second mating test (441-+32 CCK-8 vs. 575---27 saline, p<0.01, independent t-test). However, no such reduction was observed with CCK-8 in the first test of intact males, nor in the postcastration tests. Moreover, when data were analyzed using matched pairs from the crossover design, ejaculation latencies were not significantly different between CCK-8 and control treatments either before or after castration. Because CCK-8 had no effect following castration, the lack of an effect of CCK-8 on male behavior in the present experiment does not appear to be directly related to the presence of testicular hormones in adulthood, or to the normally high levels of copulatory behavior displayed by intact males which may have masked an effect. Using a higher dose of CCK-8 than we used (5 tzg/rat, IP), Linden et al. [31] also observed no effects on male copulatory behavior. Pfaus et al. [46] reported that males injected peripherally with 8 t~g/kg (subcutaneous) had a reduced ejaculation latency and intromission frequency. Since 8/zg/kg CCK-8 is a considerably higher dose than what is needed to decrease exploratory behaviors [11] and food consumption [12,30] in rats, and since ejaculation latency and intromission frequency can be reduced by "stressful" events such as operative trauma (sham castration) or audiogenic stimulation [15], perhaps the regimen used by Pfaus et al. [46] reduced these behavioral parameters by acting in some stress-related manner. Following estrogen priming, lordosis behavior was also
CCK-8 AND REPRODUCTIVE
BEHAVIORS IN MALES
353
TABLE 1 EFFECTS OF PERIPHERALLY ADMINISTERED CCK-8 (3 g.g/kg) OR SALINE (IP) ON SCORES OF MALE COPULATORY BEHAVIOR* IN INTACT AND GONADECTOMIZED MALE RATS (MEAN - SE) Days Post Castration
Treatment
(N)t
IL 57.9 --- 18 138.6 ± 64
0 (Test 1)
Saline CCK-8
(7) (8)
0 (Test 2)
Saline CCK-8
(7) (7)
3
Saline CCK-8
9
MF
IF
EL
ICI
PEI
8.7 ± 7.3 ±
1 2
15.4 _ 1 11.8 --+ 1
477 678
± 63 ± 143
59.8 --- 12.4 74.0 --+ 15.4
308 ± 52 394 ± 21
22 12
8.7 ± 11.9 ±
1 3
15.4 ± 1 11.6 ± 1.3
575 441
± 27 --- 31
39.3 ± 42.4 ±
358 ± 23 323 ± 20
(7) (7)
53.4 ± 29 148.3 ± 112
8.9 ± 9.1 ±
3.6 2.2
10.9 ± 1 8.9± 1
497 424
± 102 ± 49
52 48
± 11 ± 5
475 ± 476 ±
Saline CCK-8
(4) (6)
209.8 ± 112 255.0 -+ 163
6.3 ± 12.0 ±
3 4
7.0 ± 1 6.3 ± 0.7
293 548
± 73 ± 163
40 94
± 8 ± 35
618 ± 144 547 ± 86
13
Saline CCK-8
(5) (2)
117.8 ± 56 663.0± 605
15.2 - 8 12.5-+ 5
7.0 ± 0.7 9.0 ± 0
493 470
± 207 ± 99
66 47
± 24 --- 6
758 ± 156 578 ± 63
18
Saline CCK-8
(2) (6)
402.0 ± 293 236.0 ± 113
18.0 ± 7 25.0 -+ 9
8.0 ± 1 7.0 ± 1
1194 941
± 116 ± 263
141 205
± 22 ± 79
552 ± 10 464 --- 101
23
Saline CCK-8
(5) (1)
236.0 ± 771.0
22.0 ± 27.0
7.0 --- 1 7.0
624 918
± 206
114 131
± 61
495 ± 100 675
34
Saline CCK-8
(0) (3)
. 291.0 ±
. 9.7 ± 1
. . 884.7 ± 421
82
± 25
499 _
Saline CCK-8
(1) (0)
126
50
63.0 ± 32.6 ±
95
8
. 82
. 24.0 ± 15 20.0
.
.
14.0 .
.
1234 .
88
3.4 3.8
32 40
15
843
.
*IL=intromission latency (sec). M F = m o u n t frequency. IF=intromission frequency. EL =ejaculation latency (sec). ICI=intercopulatory interval (sec). PEI=postejaculatory interval (sec). "~Number of animals displaying ejaculatory pattern. Method n o t significantly d i f f e r e n t b e t w e e n I P C C K - 8 a n d salinei n j e c t e d g r o u p s (saline, LQ=32.3---15; C C K - 8 , L Q = 2 7 . 4 - + 18.2). T h e s e r e s u l t s d e m o n s t r a t e t h a t a n I P d o s e o f C C K - 8 t h a t d r a m a t i c a l l y a l t e r e d l o r d o s i s b e h a v i o r in g o n a d e c t o m i z e d f e m a l e s [5] w a s totally i n e f f e c t i v e in altering lord o s i s in g o n a d e c t o m i z e d m a l e s . EXPERIMENT 2 B e c a u s e t h e n u m b e r o f C C K - 8 i m m u n o r e a c t i v e cells a n d t h e t i s s u e levels o f C C K - 8 in t h e M P O , B N S T , a n d M e A are highly s e x u a l l y d i m o r p h i c w i t h g r e a t e r v a l u e s in m a l e s t h a n in f e m a l e s [20, 35, 37, 38, 49], w e w e r e r e l u c t a n t to c o n c l u d e t h a t this p e p t i d e h a s n o effect in m a l e s . T h e d o s e o f C C K - 8 m i g h t n o t h a v e b e e n sufficient to affect male c o p u l a t o r y beh a v i o r (see [46]) or the peripherally a d m i n i s t e r e d C C K - 8 might n o t h a v e r e a c h e d t h e a p p r o p r i a t e b r a i n a r e a s . S t e r n et al. [53] r e p o r t e d t h a t 3 H - c a e r u l e i n , a d e c a p e p t i d e w i t h struct u r a l a n d p h y s i o l o g i c a l h o m o l o g i e s to C C K - 8 , p r e f e r e n t i a l l y a c c u m u l a t e d in t h e V M H a f t e r p e r i p h e r a l injection. O t h e r s , however, have argued that CCK-8 administered peripherally is a c t i n g v i a p e r i p h e r a l r e c e p t o r s t h a t lie o u t s i d e t h e b l o o d b r a i n b a r r i e r [12]. I n o r d e r to t e s t f u r t h e r t h e h y p o t h e s i s t h a t C C K - 8 is a c t i n g in t h e C N S o f t h e m a l e rat to m o d u l a t e r e p r o d u c t i v e b e h a v i o r , we a d m i n i s t e r e d C C K - 8 i n t r a v e n tricularly ( I C V ) a n d studied its effects o n male c o p u l a t o r y and lordosis behaviors.
T w e n t y - o n e adult male rats w e r e p r e s e l e c t e d for male s e x u a l b e h a v i o r (see G e n e r a l M e t h o d section). O n e w e e k following p r e s e l e c t i o n tests, rats w e r e a n e s t h e t i z e d w i t h 3 ml/kg E q u i t h e s i n (0.85 g c h l o r a l h y d r a t e , 0.21 g s o d i u m p e n t o b a r b i t a l / 2 0 ml) a n d u n i l a t e r a l guide c a n n u l a e (22 ga) w e r e surgically i m p l a n t e d ; t h e tips w e r e l o c a t e d 1.0 m m a b o v e t h e lateral v e n t r i c l e , a n d c a n n u l a e w e r e a n c h o r e d w i t h skull s c r e w s . R a t s w e r e a l l o w e d o n e w e e k to r e c o v e r b e f o r e b e h a v i o r a l testing. D u r i n g i n j e c t i o n s , r a t s w e r e r e m o v e d f r o m t h e i r c a g e s a n d lightly r e s t r a i n e d . A 30 gauge wire s t y l e t t h a t w a s p r e s e n t in t h e guide c a n n u l a w a s r e m o v e d a n d rep l a c e d w i t h a n i n j e c t i o n c a n n u l a t h a t e x t e n d e d 1.0 m m bey o n d t h e guide c a n n u l a . I n j e c t i o n c a n n u l a e w e r e c o n n e c t e d with p o l y e t h y l e n e tubing to a h a n d d r i v e n microsyringe. Intrav e n t r i c u l a r i n j e c t i o n s o f 5.0/xl w e r e m a d e o v e r a o n e m i n u t e p e r i o d a n d t h e i n j e c t o r r e m a i n e d in place for a n a d d i t i o n a l 30 s e c o n d s to allow for diffusion o f solution f r o m t h e tip. I n o r d e r to verify c o r r e c t p l a c e m e n t o f c a n n u l a e , rats w e r e g i v e n a n o v e r d o s e o f a n e s t h e t i c , g i v e n a 3 . 0 / x l injection o f india ink b y i n s e r t i n g t h e i n j e c t i o n c a n n u l a into the guide c a n n u l a , t h e n p e r f u s e d i n t r a c a r d i a l l y w i t h 0.9% saline foll o w e d b y 10% n o r m a l formalin. B r a i n s w e r e c u t w i t h a r a z o r b l a d e in the c o r o n a l p l a n e , a n d p l a c e m e n t w a s verified b y observing the stained ventricular system. One rat had an i n c o r r e c t c a n n u l a p l a c e m e n t ; the d a t a f r o m this a n i m a l w e r e n o t u s e d in t h e study.
354 M a l e c o p u l a t o r y b e h a v i o r . CCK-8 was administered 10 minutes before behavioral testing. Six males received 50 ng CCK-8, eight received 500 ng CCK-8, and six were injected with the aCSF vehicle (see General Method section). Standard measures of male sexual behavior were obtained as described in the General Method section, and data were evaluated using analysis of variance and Duncan's Multiple Range Test. L o r d o s i s b e h a v i o r . Five days after the male copulatory test, the males were gonadectomized using ether anesthesia. Starting two weeks after gonadectomy, the rats were given four mating tests, each separated by 10 days. In order to safeguard against any bias in treatment, CCK-8 or a C S F treatments were assigned randomly among the animals for each test. Animals were primed with 5 tzg EB/day x 3 days for the first 3 mating tests, and the dose was increased to 30 /zg EB/day x 3 days for the fourth test in order to study the effects of CCK-8 in highly receptive males. Intraventricular injections were performed in the same manner as described for the male copulatory tests, but because of the long period of implantation, we anticipated that some cannulae assembly would eventually become loosely attached to the skull and therefore studied only 2 groups: vehicle (aCSF) and 500 ng CCK-8. Lordosis quotients were obtained as described in the General Method section, and these were evaluated by 2-way analysis of variance (treatment by testing session). Because there was a significant effect of treatment, mating tests were evaluated separately using Bonferroni's method of simultaneous inference [40]. Results and Discussion
Twenty of the 21 rats had histologically confirmed injection sites within the lateral ventricle. During the course of the experiment, the cannulae assembly in 7 of the 20 males became loosely attached to the skull. When this occurred, the animal was removed from further study. All 20 males were studied in the male copulatory test, 18 were studied in the first lordosis test, and this number was further reduced to 13 by the final lordosis test. As in Experiment 1, CCK-8 did not significantly alter any component of male sexual behavior when compared to vehicle controls. Measurements of IL, MF, ICI, and PEI were quite similar among the 0 ng, 50 ng, and 500 ng CCK-8 groups (n =6, 6, and 8, respectively). I F and E L were reduced in the 50 ng and 500 ng CCK-8 males, but individual values were quite variable and these effects were not statistically significant. (IF: 11.2-+1.5 in the 0 ng CCK-8 group, and 6.8-+1.2 and 8.3+1.6 in the 50 ng and 500 ng groups, respectively. EL: 825-+226 seconds in the 0 ng CCK-8 group, and 527-+189 and 474+14 in the 50 ng and 500 ng groups). All animals showed ejaculatory behavior during the behavioral test. Following gonadectomy and estrogen priming, animals receiving 500 ng CCK-8 ICV showed dramatically higher L Q ' s than aCSF controls. There was a significant treatment effect, F=16.94, p<0.001, but there was no significant effect of testing session and no significant treatment x testing session interaction. Comparison between CCK-8 and aCSF groups revealed that CCK-8 ICV increased L Q ' s significantly in tests 2 and 3 (Fig. 1). The results of this experiment demonstrate that injection of 50 ng or 500 ng CCK-8 ICV had no significant effects on male copulatory behavior in intact males. These results may imply that male rats are refractive to the effects of CCK-8.
BLOCH, BABCOCK, GORSKI A N D MICEVYCH
100 -
i
9080~- 7 0 z w
60-
O
D 50
o
--- 4 0
o~ 3o n~ o ~ 20
lO 0 DALLYEB DOSE:
//// ////
U// I///
8 TEST I 5#g
9 TEST 2 5~Q
. . . .
! ......
TEST 3 5~g
??5
6
TEST 4 30/Jg
FIG. 1. Effects of intracerebroventricular administration of 500 ng CCK-8 (clear bars) or artificial CSF (vehicle) on lordosis behavior in estrogen-primed males gonadectomized in adulthood. Animals were given 5 tzg EB/day x 3 days before Tests 1-3 and 30 p.g EB/day x3 days before Test 4. Each test was separated by 10 days. *p<0.02 vs. CSF treated males.
However, the dramatic increase in the LQ of gonadectomized estrogen-primed male rats (see Fig. 1) indicates that 500 ng CCK-8 is capable of affecting reproductive behavior in males. As expected, the LQ of the a C S F controls was quite high in the fourth lordosis test, which was conducted after estrogen priming with 30/zg EB. This is in agreement with previous reports showing that the LQ of gonadectomized, EB-primed males increases with repeated tests at a given dose of estrogen priming, or with higher doses of EB [15,17]. In sharp contrast with our findings in females given IP CCK-8 [5] or CCK-8 within the VMH [3], there was no indication in the present study of a reduced LQ in males after ICV injection. Indeed, under conditions of increasing behavioral receptivity the LQ was still higher in the males treated with ICV CCK-8 than in the C S F controls (Fig. 1, test 4). GENERAL DISCUSSION CCK-8 failed to alter any parameter of male copulatory behavior after peripheral (IP) injections in intact or gonadectomized male rats, or after central (ICV) injections. In sharp contrast, ICV CCK-8 significantly and dramatically increased the frequency of lordotic responses in gonadectomized, estrogen-primed males. Several lines of evidence indicate that the lack of a CCK-8 effect on male behavior is not due to an inability of ICV CCK-8 to influence the CNS. Firstly, a dose of CCK-8 that did not alter male copulatory behavior did facilitate lordosis behavior dramatically in the same males. Secondly, the 500 ng dose that we used is approximately 100 fold higher than the effective dose to inhibit L H secretion when injected within the third ventricle of the female rat [56] and between 10 and 100 times higher than the dose required to reduce LQ scores when administered within the VMH of female rats [3]. When administered ICV, the doses of CCK-8 required to suppress feeding and exploratory behaviors are higher than we used [11,49], so it is possible that the dose we used was insufficient to influence male behavior. Dornan and Malsbury [19], however, have reported that 200 ng of CCK-8 injected into the preoptic-anterior hypothalamic area of males failed to affect male copulatory behavior, indicating that injection of a relatively high dose of CCK-8 into a region
CCK-8 A N D R E P R O D U C T I V E B E H A V I O R S IN M A L E S
355
that is important for the expression of male copulatory behavior [2, 14, 24, 32, 52] was also ineffective. The dramatic facilitatory effect of ICV CCK-8 on lordosis behavior in males is especially interesting. In contrast to females, males gonadectomized in adulthood show relatively low levels of lordosis behavior following estrogen or combined estrogen/progesterone treatment [10,16]. The relatively low level of lordosis behavior in males is due to the influence of testicular hormones exerted during the neonatal period; gonadectomy during the neonatal period but not at a later time results in a male that shows a level of receptivity in adulthood equivalent to that displayed by normal females (for review, see [21]). Lordosis behavior can also be increased by experimental manipulation of males in adulthood. When estrogen is administered in high daily doses to male gonadectomized as adults, the frequency of lordotic responses approaches that of females, although much higher doses o f estrogen are needed for similar effects [16,17]. It is therefore possible that substrates exist within the male brain that influence lordosis behavior in a manner similar to that proposed for the female. Indeed, although the dose is much higher than required in a female, estrogen implants within the VMH also increase lordosis behavior in male rats [18]. As in the female, lesions of the VMH reduce lordosis behavior of the male [8] while lesions of the POA increase lordosis behavior [25]. Because lesions of the lateral septum [41,42] or anterior roof deafferentation, which cuts the connections between the septum and the preoptic area [59], also increase lordosis behavior in males, it appears that the behaviorally differentiating action of testicular hormones in the neonatal period may be due, in part, to the development of a neural system that suppresses lordosis in the normal male [25]. We have shown that CCK-8 itself will not induce lordosis behavior in females [5] and males (unpublished observation) but modifies only an estrogen-primed system. Thus, the relatively rapid effects of CCK-8, occurring within 10 minutes following its administration ICV, could be exerted through an interaction with cells in one or more areas of the brain, but only after estrogen has been allowed to initiate de novo synthesis of proteins or other substrates essential for lordosis behavior [28, 34, 58]. Because increased sexual receptivity in estrogen-primed female rats can be accomplished by giving progesterone or " p r o g e s t e r o n e substitutes" that include cholinergic agonists, serotonergic agonists and antagonists, dopaminergic agonists and antagonists, ovarian and adrenal hormones, and a variety of peptides (see [13, 27, 57]), this has led to speculation that a "nonspecific" mechanism may be involved [57]. However, the male rat differs from the female in that lordosis behavior in the estrogen-primed normal [10,17] or septal-lesioned [41] male is not further increased by progesterone. Moreover, the facilitation of lordosis behavior following peripheral administration of CCK-8 in lowreceptive, ovariectomized, estrogen-primed female rats is also observed after adrenalectomy, indicating that progesterone is not required for this effect in females [5]. Thus, it appears unlikely that CCK-8 would be acting on this system in the male. We have proposed that CCK-8 is acting within the hypothalamus of the female to inhibit the effects on lordosis behavior that are attributed to that site [3]. The regions of the
hypothalamus that appear to be most important for the regulation of the lordosis behavior in estrogen-primed female and male rats are the VMH and MPO [7, 8, 25, 43, 45, 47]. Lordosis behavior of the female rat appears to be under the dual control of these regions. According to this concept, estrogen stimulates cells of the V M H to increase lordosis behavior of the female and simultaneously estrogen acts in the POA to remove preoptic inhibition of this behavior [4,7]. Thus, the V M H is a facilitatory site for lordosis behavior in the estrogen-primed female (see [43] for review), and CCK-8 could be acting to inhibit this action and thereby attenuate this behavior. Similarly, CCK-8 could be acting to inhibit the inhibitory action of the MPO on lordosis behavior in the female [7, 45, 47] and thereby increase this behavior. As discussed previously [3], this model can explain the types of complex results in the female that we have reported after peripheral administration of CCK-8 [5]. In the estrogenprimed female rat, CCK-8 administered into the V M H decreases lordosis behavior [3], while preliminary results in our laboratory indicate that CCK-8 administered into the MPO facilitates this behavior (Dornan, Bloch and Micevych, unpublished). In the present experiment, lordosis behavior was not reduced in males administered CCK-8 ICV under conditions of high receptivity, a result that we have observed after peripheral [5] or VMH [3] administration of this peptide in females. One explanation for this difference in response to CCK-8 between males and females is that the ICV administered CCK-8 in the present study did not perfuse the VMH whereas the peripheral (and VMH) application in females did reach this brain area. Indeed, peripherally administered CCK-8 and injections of CCK-8 into the VMH may be acting on the same site [53]. A more likely explanation is that the CCK-sensitive neural circuits which regulate lordosis behavior are sexually dimorphic. There are several sex differences within the regions of the hypothalamus and limbic system that contain CCK-8 (see Introduction). We are therefore testing the possibility that the neural substrate responsible for the initiation of lordosis behavior is different in males and females. In the estrogen-primed male, preliminary results indicate that, as in the female, CCK-8 administered into the MPO has a facilitatory effect on lordosis behavior (Bloch et al., unpublished), and we are also studying males that will receive CCK-8 in the VMH. By comparing the doses of CCK-8 that are needed to produce alterations of lordosis behavior in the male and the female, we will test the hypothesis that there are site-specific sex differences in the lordosis response to CCK-8. In summary, we have demonstrated that peripheral administration of a dose of CCK-8 that is effective in altering lordosis behavior in females does not alter either male or female reproductive behavior in males. Although central administration of CCK-8 also did not affect male copulatory behavior, lordosis behavior was dramatically increased in these males after gonadectomy and estrogen priming. These results indicate that exogenous CCK-8 is acting in the male either to inhibit a neural system that normally suppresses lordosis behavior, or is acting directly on neural substrates that facilitate lordosis behavior. In addition, these results imply that there may be a significant sex difference in how CCK-responsive circuits are organized within the brain to regulate lordosis behavior.
356
BLOCH, BABCOCK, GORSKI AND MICEVYCH
REFERENCES 1. Akesson, T. R.; Mantyh, P. W.; Mantyh, C. R.; Matt, D. W.; Micevych, P. E. Estrous cyclicity of 125-1-CCK octapeptide binding in the ventromedial nucleus: evidence for down modulation by estrogen. Neuroendocrinology 45:257-262; 1986. 2. Arendash, G. W.; Gorski, R. A. Effects of discrete lesions of the sexually dimorphic nucleus of the preoptic area or other medial preoptic regions on the sexual behavior of male rats. Brain Res. Bull. 10:147-154; 1983. 3. Babcock, A. M.; Bloch, G. J.; Micevych, P. E. Injections of cholecystokinin into the ventromedial hypothalamic nucleus inhibit lordosis behavior in the rat. Physiol. Behav., in press; 1988. 4. Bartield, R. J.; Chen, J. J. Activation of estrous behavior in ovariectomized rats by intracerebral implants of estradiol benzoate. Endocrinology 101:1716-1725; 1977. 5. Bloch, G. J.; Babcock, A. M.; Gorski, R. A.; Micevych, P. E. Cholecystokinin stimulates and inhibits lordosis behavior in female rats. Physiol. Behav. 39:217-224; 1987. 6. Bloch, G. J.; Davidson, J. M. Effects of adrenalectomy and experience on postcastration sex behavior in the male rat. Physiol. Behav. 3:461-465; 1968. 7. Bueno, J.; Pfaff, D. W. Single unit recording in hypothalamus and preoptic area of estrogen-treated and untreated ovariectomized female rats. Brain Res. 101:67-78; 1976. 8. Chateau, D.; Chabli, A.; Aron, C. Effects of ventromedial nucleus lesions on the display of lordosis behavior in the male rat. Interactions with facilitory effects of male urine. Physiol. Behav. 39:341-345; 1987. 9. Christenson, L. W.; Nance, D. M.; Gorski, R. A. Effects of hypothalamic and preoptic lesions on reproductive behavior in male rats. Brain Res. Bull. 2:137-141; 1977. 10. Clemens, L. G.; Shryne, J.; Gorski, R. A. Androgen and development of progesterone responsiveness in male and female rats. Physiol. Behav. 5:673-678; 1970. 11. Crawley, J. N.; Hays, S. E.; O'Donahue, T. L.; Paul, S. M.; Goodwin, F. K. Neuropeptide modulation of social and exploratory behaviors in laboratory rodents. Peptides 2:Suppl 1, 123129; 1981. 12. Crawley, J. N.; Rojas-Ramirez, J. A.; Mendelson, W. B. The role of central and peripheral cholecystokinin in mediating appetitive behaviors. Peptides 3:535-538; 1982. 13. Crowley, W. R.; Zemlan, F. P. The neurochemical control of mating behavior. In: Adler, N. T., ed. Neuroendocrinology of reproduction. New York: Plenum Press, 1981:451-484. 14. Davidson, J. M. Activation of the male rat's sexual behavior by intracerebral implantation of adrogen. Endocrinology 79:783794; 1966. 15. Davidson, J. M. Hormonal control of sexual behavior in adult rats. In: Raspe, G., ed. Advances in the bioscieneces. (Schering symposium on endocrinology). New York: Pergamon Press, 1967:11%141. 16. Davidson, J. M. Effects of estrogen on the sexual behavior of male rats. Endocrinology 84:1365--1372; 1969. 17. Davidson, J. M.; Bloch, G. J. Neuroendocrine aspects of male reproduction. Biol. Reprod. 1:67-92; 1969 18. Davis, P. G.; Barfield, R. J. Activation of feminine sexual behavior in castrated male rats by intrahypothalamic implants of estradiol benzoate. Neuroendocrinology 28:228-233; 1979. 19. Dornan, W. A.; Malsbury, C. W. The effects of medial preoptic area injections of substance P and cholecystokinin on male rat sexual behavior. Ann. Conf. Sex. Behav. 19:43; 1987. 20. Frankfurt, M.; Siegel, R. A.; Sim, I.; Wuttke, W. Cholecystokinin and Substance P concentrations in discrete areas of the rat brain: sex differences. Brain Res, 358:53-58; 1985. 21. Gorski, R. A. The neuroendocrine regulation of sexual behavior. In: Newton, G.; Riesen A. H., eds. Advances in psychobiology. New York: John Wiley & Sons, 1974:1-58.
22. Gorski, R. A.; Harlan, R. E.; Jacobson, C. D.; Shryne, J. E.; Southam, A. M. Evidence for the existence of a sexually dimorphic nucleus in the preoptic area of the rat. J. Comp. Neurol. 193:52%539; 1980. 23. Harris, V. S.; Sachs, B. D. Copulatory behavior in male rats following amygdaloid lesions. Brain Res. 86:514-518; 1975. 24. Heimer, L. ; Larsson, K. Impairment of mating behavior in male rats following lesions of the preoptic-anterior hypothalamic continuum. Brain Res. 3:248-263; 1966/1967. 25. Hennessey, A. C.; Wallen, K. ; Edwards, D. A. Preoptic lesions increase the display of lordosis by male rats. Brain Res. 370: 21-28; 1986. 26. Hines, M.; Davis, F. C.; Coquelin, A.; Goy, R. W.; Gorski, R. A. Sexually dimorphic regions in the medial preoptic area and the bed nucleus of the stria terminalis of the guinea pig brain: a description and an investigation of their relationship to gonadal steroids in adulthood. J. Neurosci. 5:40-47; 1985. 27. Hunter, A. J.; Hole, D. R.; Wilson, C. A. Studies into the dual effects of serotonergic pharmacological agents on female sexual behavior in the rat: Preliminary evidence that endogenous 5HT is stimulatory. Pharmacol. Biochem. Behav. 22:5-13, 1985. 28. Jones, K. J.; Chikaraishi, D. M.; Harrington, C. A.; McEwen, B. S.; Pfaff, D. W. In situ hybridization detection of estradiolinduced changes in ribosomal RNA levels in rat brain. Mol. Brain Res. 1:145-152; 1986. 29. Kimura, F.; Hashimoto, R.; Kawakami, M. The stimulatory effect of cholecystokinin implanted in the medial preoptic area on luteinizing hormone secretion in the ovariectomized estrogen-primed rat. Endocrinol. Jpn. 30:305-309; 1983. 30. Kraly, F. S. A diurnal variation in the satiating potency of cholecystokinin in the rat. Appetite: J. Intake Res. 2: 177-191; 1981. 31. Linden, A.; Hansen, S.; Bednar, I.; Forsberg, G.; Srdersten, P. ; Uvnas-Moberg, K. Sexual activity increases plasma concentrations of cholecystokinin octapeptide and offsets hunger in male rats. J. Endocrinol. 115:91-95; 1987. 32. Lisk, R. D. Copulatory ability of the male rat following placement of medial preoptic-anterior hypothalamic lesions. Exp. Brain Res. 5:306-313; 1968. 33. Matsumoto, A.; Arai, Y. Sex difference in volume of the ventromedial nucleus of the hypothalamus in the rat. Endocrinol. Jpn. 30:277-280; 1983. 34. Meisel, R. L.; Pfaff, D. W. RNA and protein synthesis inhibitors: Effects on sexual behavior in female rats. Brain Res. Bull. 12:187-193; 1984. 35. Mendelson, S. D.; Gorzalka, B. B. Cholecystokinin-octapeptide produces inhibition of lordosis in the female rat. Pharmacol. Biochem. Behav. 21:755-759; 1984. 36. Micevych, P. E.; Akesson, T. R.; Elde, R. P. The distribution of cholecystokinin immunoreactive cell bodies in the male and female rat. II. Bed nucleus and amygdaloid complex. J. Comp. Neurol., in press; 1988. 37. Micevych, P. E.; Babcock, A. M.; Matt, D. W.; Lu, J. K. H. Cholecystokinin inhibits k+ stimulated LHRH release from male and female rat hypothalami in vitro. Soc. Neurosci. Abstr. 12:1415; 1986. 38. Micevych, P. E.; Matt, D. W.; Go, V. W. Brain distribution of CCK, sP and bombesin in male and female rats during the estrous cycle. Exp. Neurol., in press; 1988. 39. Micevych, P. E.; Park, S. S.; Akesson, T. R.; Elde, R. Distribution of cholecystokinin-immunoreactive cell bodies in the male and female rat: I. Hypothalamus. J. Comp. Neurol. 255:124-136; 1987. 40. Moses, L. E. Think and explain with statistics. Reading, MA: Addison-Wesley; 1986. 41. Nance, D. M.; Shryne, J.; Gorski, R. A. Septal lesions: effects on lordosis behavior and pattern of gonadotropin release. Horm. Behav. 5:73-81; 1974.
CCK-8 AND REPRODUCTIVE
BEHAVIORS IN MALES
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